Journal: Frontiers in Immunology

Article Title: Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo

doi: 10.3389/fimmu.2023.1226518

Figure Lengend Snippet: Generation of NK-92-CAR.19-IL-15/IL15Rα cells. (A) Scheme of CAR constructs targeting CD19 (CAR.19) under the control of spleen focus-forming virus (SFFV) promoter. CAR consists of scFv, CD8 hinge and transmembrane region, 41BB costimulatory molecule and CD3ζ signaling molecule. Where indicated, the CAR sequences are followed by a self-cleaving peptide (T2A) and IL-15 or IL-15/IL-15Rα (IL-15 and IL-15Rα fused through a flexible linker). (B, C) NK-92 cells were transduced with the described CAR constructs followed by immunomagnetic enrichment in two steps. (B) Representative dot plots show flow cytometric analysis of CAR expression on freshly transduced and enriched NK-92-CAR.19 cells. (C) The graph shows the frequency of CAR-positive cells before selection, after first and second selection, and 30 days after second selection.

Article Snippet: Supernatants were harvested and IL-15, IFN-γ and TNFα were measured by multiplex assay using the MILLIPLEX MAP Human CD8+ T-Cell magnetic Bead Panel kit and MILLIPLEX Human Cytokine/Chemokine/Growth factor Panel A (Merck-Millipore).

Techniques: Construct, Control, Virus, Transduction, Expressing, Selection

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Epitope-specific expansion cultures (ESPEC) efficiently and specifically expand desired major histocompatibility complex (MHC) I-restricted and MHC II-restricted T cell responses. ( A ) Overview of workflow for ESPEC; ( B ) overview of analyzed patients and antigens used for ESPEC-SUIT; ( C ) frequency of antigen-specific T cell responses as determined by interferon (IFN)γ Enzyme-Linked ImmunoSpot (ELISpot) in peripheral blood mononuclear cell (PBMC) ( ex vivo , gray, 300 000 cells/well) or post-ESPEC samples (colored, 25 000–50 000 cells/well) for patients with glioma undergoing vaccination with shared (neo-1, n=22; neo-2, n=5) or individual MHC II-restricted neoepitopes (i1-3, n=2 patients, n=3 antigens); open symbols (gray/colored) indicate lack of antigen-specific T cell response ( ex vivo or post-ESPEC); raw data inlays show well images of a representative neo-1 vaccinated patient post-ESPEC for no peptide (‘no pep’) and neo-1-stimulated cultures (top) versus ex vivo (bottom), re-stimulated without antigen (ns) or with neo-1 peptide; ( D ) frequency of antigen-specific T cell responses determined by IFNγ ELISpot in post-ESPEC samples (100 000–250 000 cells/well) for patients with liposarcoma ( i4 ), melanoma ( i5 ) or colorectal cancer ( i6 ) not undergoing vaccination at time of blood sampling. i4 and i6 were in the past vaccinated with long peptides encompassing mutations also contained in short (9–11-mers, MHC I-presented) or long (>20 mer, MHC II-presented) peptides used in ESPEC (‘targeted’, dark gray). Long peptides used for stimulation contained amino acid sequences spanning point mutations or resulting from fusions/missense mutations; short peptides were mutation-derived epitopes predicted to bind the patient’s MHC I molecules; ESPEC also included antigens not targeted by vaccination (‘non-targeted’, light gray); numbers above datasets indicate no. of antigens investigated; heatmap of IFNγ ELISpot spot-forming units (SFU) in ( E ) cultures stimulated with patient-individual ( i6 ) 20-mer peptides (x-axis) after re-stimulation (y-axis) without antigen (‘no peptide’), with the antigen-of-interest (diagonal) or control antigen (MOG), or ( F ) healthy donor (HD1)-derived cultures (x-axis) after ESPEC with four different peptide pools (pp1–pp4) of short (MHC I-restricted, ‘( I )’) or long (MHC II-restricted, ‘(II)’) recall antigens. Post-ESPEC, cells were re-stimulated (y-axis) with single antigens present (circles in heatmap) or absent from the pool; schematic graph to left of heatmap shows strength of the response to the antigens when used as single peptides in ESPEC; intracellular cytokine staining of tumor necrosis factor (TNF)α production in living/CD3 + /CD4 + T cells after re-stimulation of 1×10 6 ( ex vivo ) or 0.4×10 6 (post-ESPEC) cells/condition in representative patients undergoing vaccination with ( G ) neo-1 or ( H ) two patient-individual neoepitopes (neo-X, neo-Y). Panels show the response pre-ESPEC (‘ ex vivo’ ), and post-ESPEC in control stimulated cultures (‘no peptide’) or cultures stimulated with antigen-of-interest (‘neo-1/X/Y’, as indicated above the panel), gated on CD4 + T cells (ID15 and i1-2 top panel) vs CD8 + T cells (i1-2, bottom panel). Histograms show responses to re-stimulation with various peptides: CMV (cytomegalovirus) peptide is included as a control for the unspecific expansion of endogenous T cell responses, wt-1 is included to assess cross-reactivity of neo-1-stimulated cells and neo-X/Y are specificity controls for recently stimulated T cells. APC, antigen-presenting cell; TCR, T cell receptor; neo, neoepitope.

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: Immunopeptidomics, Enzyme-linked Immunospot, Ex Vivo, Sampling, Mutagenesis, Derivative Assay, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Epitope-specific expansion cultures (ESPEC)-induced changes in T cell receptor (TCR) repertoire composition. TCR repertoire ( A ) diversity (mean±SD) and ( B ) clonality (mean±SD) of post-ESPEC samples (n=80 from 32 individuals) expanded without peptide (‘ctrl’) or under antigen-of-interest (‘stim’); note that some donors were stimulated with multiple different peptides or peptide pools in separate cultures, thus contributing multiple post-ESPEC samples and a single ‘ctrl’. # **p≤0.01, Mann-Whitney U test; two-tailed; ( C ) mean percentage of repertoire occupied by clones with rank 1–100, 101–1000 or 1001–10 000 in post-ESPEC samples expanded without peptide (‘ctrl’, n=32) or with antigen (‘stim’, n=80, as some donors were stimulated with multiple antigens) # ; ( D ) clonality in antigen-of-interest (‘stim’) or control stimulated cultures (‘ctrl’), where ESPEC induced an increase in the ELISpot response to neo-1 stimulation of <10-fold (n=7) or >10-fold (n=11) # ; ( E ) fold expansion of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC frequency in two healthy donors (HD2, HD3) and patient i4 stimulated with MHC I-restricted (light gray) or MHC II-restricted (dark gray) recall antigens (top panel), and three patients ( i3–i4, i6 ) stimulated with short (light gray) or long (dark gray) peptides encoding patient-individual neoepitopes, including a patient-individual gene fusion targeted by peptide vaccination ( i3 ). Full repertoire data were evaluated to avoid loss of small clones by down-sampling. Numbers next to the y-axis indicate number of top 10 post-ESPEC clonotypes that were below the limit of detection pre-ESPEC; ( F ) fold expansion of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC frequency in all neoepitope ESPEC cultures (n=63), sorted by antigen type and magnitude of the post-ESPEC ELISpot response # . Symbol hues indicate cultures enriched for CD4 + (teal, mint, light green) or CD8 + (dark green) T cells prior to repertoire sequencing. Numbers above the x-axis indicate the number of top 10 post-ESPEC clonotypes that were below the limit of detection pre-ESPEC; frequency of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC (baseline (‘BL’)) frequency for representative cultures exposed to ( G ) short and ( H ) long peptides and enriched for the relevant T cell subset # . **P≤0.01, Wilcoxon signed-rank test, two-tailed). # TCRβ deep-sequencing data were down-sampled to 10 000 TCR counts (panel A–D, F–H ).

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: MANN-WHITNEY, Two Tailed Test, Clone Assay, Control, Enzyme-linked Immunospot, Sampling, Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Tracing and characterization of antigen-reactive clonotypes in peripheral blood and tissue. ( A–B ) Representative data of the cumulative frequency of epitope-specific expansion cultures (ESPEC)-derived candidate T cell receptors (TCRs) (bars, left y-axis) and the ex vivo interferon (IFN)γ Enzyme-Linked ImmunoSpot (ELISpot) responses against neo-1 peptide (background subtracted, dotted line, right y-axis) at the relevant time points in peripheral blood of two neo-1 vaccinated patients. In ( B ), clonotypes with in vitro validated neo-1 reactivity are highlighted in teal, untested or unreactive candidates are shown in gray; for comparison between peripheral blood mononuclear cell (PBMC) samples, TCRβ deep-sequencing data was down-sampled to 50 000 counts; blood was collected before vaccination and after 1–7 administrations of long neo-1 encoding peptide (A: vaccination on week ( W ) 0, 2, 4, 10; B: vaccination on W0, 2, 4, 9, 17); number ( C ) and cumulative frequency ( D ) of ESPEC-derived candidate clonotypes (teal) versus all other clonotypes in tumors of neo-1 immunized patients (n=4, 5 tumors), and patient i3 (two tumors), who received a personalized, fusion-spanning peptide vaccine; tumor tissue was collected after eight vaccinations; raw ( E ) and quantified (mean±SD) ( F ) neo-1-specific IFNγ ELISpot response in minimally cultured tumor-infiltrating lymphocytes from two patients with glioma treated with neo-1 long peptide vaccine, after stimulation of 5000 cells/well with no peptide (ctrl) or neo-1 20-mer, and ( G ) cumulative frequency of neo-1 ESPEC-derived candidate clonotypes detected in the tumor-infiltrating lymphocyte (TIL) TCR repertoire of these patients as assessed by bulk (ID7) or single-cell (ID8) TCR sequencing; uniform manifold approximation and projection highlighting gene expression profiles of tumor-infiltrating lymphocytes in n=3 neo-1 vaccinated patients with TCRβ CDR3 sequences ( H ) derived from in vitro validated neo-1-reactive clonotypes (n=70 in n=827 cells) or ( I ) shared (‘candidates’, n=246 clonotypes in n=1270 cells) and not shared (‘other’, n=6519 clonotypes in n=11 779 cells) with ESPEC candidates selected from neo-1-stimulated cultures of the respective patient.

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: Derivative Assay, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Comparison, Sequencing, Cell Culture, Gene Expression

Journal: Scientific Reports

Article Title: Cocaine-mediated impact on HIV infection in humanized BLT mice

doi: 10.1038/srep10010

Figure Lengend Snippet: Splenocytes from the sacrificed mice were also used for flow cytometric analysis of lymphocyte phenotype. The COCAINE-HIV group of mice has higher levels of ( a ) CD4CD38 and ( b ) CD8CD38. ( c ) To assess functionality of CD8 T cells, we stimulated ex vivo splenocytes in the presence of GolgiPlug to measure levels of IFN-γ production. The COCAINE-HIV group had comparable levels to HIV despite the higher numbers of CD8CD38 T cells. For all figures, *p < 0.05, **p < 0.01, using Mann-Whitney or Kruskal-Wallis test (for 3 or more groups).

Article Snippet: CD8+ T cells were isolated by magnetic bead isolation (EasySep Human CD8+ Selection Kit, Stem Cell Technologies) from the PBMC of healthy donors obtained by the UCLA CFAR Virology Core and activated overnight in 50 ng/ml anti-CD3 (clone OKT3, Imgenex, San Diego, CA) and 300 U/ml IL-2 (NIH AIDS Reagent Program).

Techniques: Ex Vivo, MANN-WHITNEY

Journal: bioRxiv

Article Title: Estrogens increase cancer cell efferocytosis to establish an immunosuppressive tumor microenvironment

doi: 10.1101/2024.12.26.630419

Figure Lengend Snippet: Expression of CX3CR1 in intratumoral TAMs from LLC1 tumors injected in ovariectomized mice treated ± E2 (n=7-8 mice/group) (A). Quantification of mean fluorescent intensity of TAM intrinsic CX3CR1 from ovariectomized Esr1 f/f and Esr1 f/f ; LysMCre mice treated ± E2 and injected with LLC1- mCherry-spectrin cells (n=5-8 mice/group) (B). Syngeneic tumor growth of LLC1 (NSCLC) (n=9-10 mice/group) (C); A7C11 (breast carcinoma) (n=10 mice/group) (D) and B16F10 (melanoma) (n=7-9 mice/group) cells in ovariectomized CX3CR1 +/gfp -HET and CX3CR1 gfp/gfp -KO (null) mice treated ± E2 (E). Quantification of GFP+ TAMs (i); CD206+ TAMs(ii) and GZMB+ CD8+ T (iii) cells from experiment described in (F). Quantification of LLC1 tumor volumes when LLC1 tumors were co-mixed with BMDM from CX3CR1 +/gfp -HET and CX3CR1 gfp/gfp -null mice and injected in ovariectomized CD45.1 mouse ± E2 (n=10 mice/group) (G). Schematic representation and quantitative PCR expression of ISGs in sorted CD11b+ GFP+ myeloid cells from LLC1 tumors injected in ovariectomized CX3CR1 +/gfp HET and CX3CR1 gfp/gfp -null mouse ± E2 (n=6-8 mice/group) (H (i) and (ii)). Quantification of IFNγ+(I) and GZMB+ (J) CD8+ T cells when T cells were cocultured with BMDM from CX3CR1 +/gfp -het and CX3CR1 gfp/gfp -null macrophages treated ± E2 (72hrs) in presence or in absence of apoptotic Jurkat cells (last 24 hrs) (n=3 mouse/condition). Data represent mean ± S.E.M. Significance is calculated by unpaired Students t-test (A, F(i)), one-way ANOVA, and pairwise comparison followed by Sidak’s multiple comparisons (I, J and K), Dunnett’s multiple comparisons (F (ii), (iii) and G) (ii)), Bonferronni’s multiple comparisons (B) by or two-way ANOVA followed by Tuckey’s multiple corrections (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

Article Snippet: CD8+ T cells were isolated from the spleens of C57BL/6J or mice with magnetic bead-based CD8 T cell isolation kit (Cat # 19853, StemCell Technologies).

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Estrogens increase cancer cell efferocytosis to establish an immunosuppressive tumor microenvironment

doi: 10.1101/2024.12.26.630419

Figure Lengend Snippet: Syngeneic tumor growth of LLC1 (NSCLC) ( A ), A7C11 (breast carcinoma) ( B ) and B16F10 (melanoma) ( C ) injected in ovariectomized C57BL6/J mouse treated with E2 in presence or in the absence of CX3CR1i AZD8797 (A-C). Flow cytometry quantification of intratumoral M1 macrophages (MHCII+) ( D ) and CD44+CD69+ ( E ) IFNγ+( F ) GZMB+ ( G ) and PD1+ ( H ) CD8+ T cells from experiment in 4B. Data represent mean ± S.E.M. Significance is calculated by Students t- test, two-way ANOVA followed by Bonferronni’s multiple corrections. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: CD8+ T cells were isolated from the spleens of C57BL/6J or mice with magnetic bead-based CD8 T cell isolation kit (Cat # 19853, StemCell Technologies).

Techniques: Injection, Flow Cytometry

Journal: bioRxiv

Article Title: Estrogens increase cancer cell efferocytosis to establish an immunosuppressive tumor microenvironment

doi: 10.1101/2024.12.26.630419

Figure Lengend Snippet: Schematic of combination treatment using radiation (5Gy) and SERD fulvestrant (A). Syngeneic tumor growth of LLC1 cells in ovariectomized mice treated with placebo, placebo+5Gy radiation, E2 + 5Gy radiation and E2+radiation+fulvestrant (25mg/kg) (n=10mice/group) ( B ). Flow cytometry quantification of CD206+ macrophages ( C ); CX3CR1+ macrophages ( D ); mCherry+ macrophages ( E ), IFNg+ CD8+ T cells ( F ); PD1+ CD8+T cells ( G ) and IFNg+CD4+ T cells ( H ) from tumors isolated from experiment described in 6B. Schematic of combination treatment using radiation and anti-CX3CR1i AZD8797 ( I ) Syngeneic tumor growth of LLC1 cells in ovariectomized mice treated with placebo; placebo+radiation (5Gy); E2+radiation (5Gy); E2+radiation+CX3CR1i (2mg/kg) n=10 animal/group ( J ). Data represents S.E.M. Significance was calculated by two-way ANOVA and pairwise comparison followed by Dunnett’s multiple corrections (B and J), one-way ANOVA and pairwise comparison followed by Dunnett’s multiple corrections (C, D, F, G and H), pairwise comparison followed by Sidak’s multiple comparisons (E) and (J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Schematics in A and I were created by biorender.com.

Article Snippet: CD8+ T cells were isolated from the spleens of C57BL/6J or mice with magnetic bead-based CD8 T cell isolation kit (Cat # 19853, StemCell Technologies).

Techniques: Flow Cytometry, Isolation, Comparison